This invention pertains to an isolated composition and a method of treating arthritis or lameness in a subject. More specifically, the invention pertains to specific growth hormone releasing hormone (“GHRH”) compositions, and methods of use thereof. The GHRH is an isolated composition or an isolated nucleic acid molecule that encodes the GHRH or functional biological equivalent. Another aspect of the current invention includes a method for delivering the composition of this invention to a subject for treating arthritis or lameness in a subject, such as a horse or other animal having arthritis or lameness.
Arthritis: The prevalence of arthritis is high, with osteoarthritis being one of the most frequent disorders in the population. In 1997, approximately 16% of the US population had some form of arthritis. This prevalence is expected to increase in the coming years, as arthritis more often affects the elderly, a proportion of the population that is increasing. The economic burden of such musculoskeletal diseases is also high, accounting for up to 1-2.5% of the gross national product of western nations. This burden comprises both the direct costs of medical interventions and indirect costs, such as premature mortality and chronic and short-term disability. The impact of arthritis on quality-of-life indicators is of particular importance. Musculoskeletal disorders are associated with some of the poorest quality-of-life indicators, particularly in terms of bodily pain. For example, bodily pain and physical functioning due to musculoskeletal disorders have mean quality-of-life indicator scores consisting of 52.1 and 49.9, respectively (values were derived from the MOS 36-item Short Form Health Survey, wherein low scores tend to indicate a limiting physical, psychic and relational health aspects of a patient and higher scores tend to indicate non-limiting aspects). In comparison to musculoskeletal disorders, the quality-of-life scale for gastrointestinal conditions is less limiting (e.g. bodily pain 52.9 and physical functioning 55.4). Other examples include: chronic respiratory diseases (e.g. bodily pain 72.7 and physical functioning 65.4); and cardiovascular conditions (e.g. bodily pain 64.7, and physical functioning 59.3) (Reginster, 2002).
Joint disease is a significant social and economic problem that needs continued research improvements for therapeutics. Pain associated with arthritis is very common throughout the world and is an increasing problem in the ageing population (Moore, 2002). Because horses have osteoarthritis conditions that are similar to human osteoarthritis conditions, the horse can be chosen as a species to investigate gene transfer as a potential therapeutic modality for the treatment of osteoarthritis (Frisbie and McIlwraith, 2000). Many compounds are being investigated for the control of symptoms of osteoarthritis in people and animals. Ideally, treatment should include analgesia, inflammation control, and chondroprotection. Currently available treatments may include: lavage of the affected joints if septic, intra-articular administration of antibiotics, hyaluronidase (e.g. Legend®, Bayer, drug used in horses) or corticosteroids, arthroscopic debridement with or without partial synovectomy, systemic administration of antibiotics, anti-inflamatory or chondroprotective drugs (Fubini et al., 1999; Murray et al., 1998; Steel et al., 1999). With further progress in this area, combination therapies tailored to the needs of the individual animal should enable us to maximize efficacy and minimize side effects. Only a few of the newer therapies and pharmaceutical agents have been investigated in the horse as a model for human arthritis, however arthritis therapies that employ biological agents are currently limited by possible side effects such as the occurrence or reemergence of viral and bacterial infections as well as their exorbitant expense (Malone, 2002). The need for a comprehensive therapy for both the joint problems and general health and welfare of the animal is critical (Naughton and Shumaker, 2003).
Growth Hormone Releasing Hormone (“GHRH”) and Growth Hormone (“GH”) Axis: To better understand utilizing GHRH plasmid mediated gene supplementation as a treatment of arthritis, the mechanisms and current understanding of the GHRH/GH axis will be addressed. Although not wanting to be bound by theory, the central role of growth hormone (“GH”) is controlling somatic growth in humans and other vertebrates. The physiologically relevant pathways regulating GH secretion from the pituitary are fairly well known. The GH production pathway is composed of a series of interdependent genes whose products are required for normal growth. The GH pathway genes include: (1) ligands, such as GH and insulin-like growth factor-I (“IGF-I”); (2) transcription factors such as prophet of pit 1, or prop 1, and pit 1: (3) agonists and antagonists, such as growth hormone releasing hormone (“GHRH”) and somatostatin (“SS”), respectively; and (4) receptors, such as GHRH receptor (“GHRH-R”) and the GH receptor (“GH-R”). These genes are expressed in different organs and tissues, including the hypothalamus, pituitary, liver, and bone. Effective and regulated expression of the GH pathway is essential for optimal linear growth, as well as homeostasis of carbohydrate, protein, and fat metabolism. GH synthesis and secretion from the anterior pituitary is stimulated by GHRH and inhibited by somatostatin, both hypothalamic hormones. GH increases production of IGF-I, primarily in the liver, and other target organs. IGF-I and GH, in turn, feedback on the hypothalamus and pituitary to inhibit GHRH and GH release. GH elicits both direct and indirect actions on peripheral tissues, the indirect effects being mediated mainly by IGF-I.
Several studies in different animal models and human have shown that GHRH has an immune stimulatory effect, both through stimulation of the GH axis and directly as an immune-modulator (Dialynas et al., 1999; Khorram et al., 2001). GH has been known to enhance immune responses, whether directly or through the IGF-I, induced by GH. Recently, a GH secretagogue (“GHS”), was found to induce the production of GH by the pituitary gland, but also determined a statistically significant increase in thymic cellularity and differentiation in old mice. When inoculated with a transplantable lymphoma cell line, EL4, the treated old mice showed statistically significant resistance to the initiation of tumors and the subsequent metastases. Generation of CTL to EL4 cells was also enhanced in the treated mice, suggesting that GHS has a considerable immune enhancing effect (Koo et al., 2001). The immune function is also modulated by IGF-I, which has two major effects on B cell development: potentiation and maturation, and as a B-cell proliferation cofactor that works together with interlukin-7 (“IL-7”). These activities were identified through the use of anti-IGF-I antibodies, antisense sequences to IGF-I, and the use of recombinant IGF-I to substitute for the activity. There is evidence that macrophages are a rich source of IGF-I. The treatment of mice with recombinant IGF-I confirmed these observations as it increased the number of pre-B and mature B cells in bone marrow (Jardieu et al., 1994). The mature B cell remained sensitive to IGF-I as immunoglobulin production was also stimulated by IGF-I in vitro and in vivo (Robbins et al., 1994).
In aging mammals, the GHRH-GH-IGF-I axis undergoes considerable decrement having reduced GH secretion and IGF-I production associated with a loss of skeletal muscle mass (sarcopenia), osteoporosis, arthritis, increased fat deposition and decreased lean body mass (Caroni and Schneider, 1994; Veldhuis et al., 1997). It has been demonstrated that the development of these changes can be offset by recombinant GH therapy. It has also been shown in culture, in vitro that the production of hyaluronan and condroitin sulphate proteoglycans is regulated by GH, IGF-I, and that these molecules may be of significant importance in the therapy of joint pathology (Erikstrup et al., 2001; Pavasant et al., 1996). For instance, gene transfer of IGF-I into rabbit knee joints promotes proteoglycan synthesis without significantly affecting inflammation or cartilage breakdown, or adverse effects. As a result, local gene transfer of IGF-I to joints was suggested as a therapeutic strategy to stimulate new matrix synthesis in both rheumatoid arthritis and osteoarthritis (Mi et al., 2000). It has been also shown that increased levels of IGF-binding proteins in arthritis may result in the reduced availability of free IGFs that can bind to IGF receptors. The observed changes in the IGF system may thus participate in the catabolic processes in rheumatoid arthritis, and the development of cachexia and wasting in these patients (Neidel, 2001). A therapy that would address both the arthritic disease and the wasting would be a major step forward in the well-being and quality of life of patients.
The production of recombinant proteins in the last 2 decades provided a useful tool for the treatment of many diverse conditions. For example, GH has been used successfully in GH-deficiencies in short stature children, or as an anabolic agent in burn, sepsis, and AIDS patients. However, resistance to GH action has been reported in malnutrition and infection. Clinically, GH replacement therapy is used widely in both children and the elderly. Current GH therapy has several shortcomings, however, including frequent subcutaneous or intravenous injections, insulin resistance and impaired glucose tolerance (Rabinovsky et al., 1992); children are also vulnerable to premature epiphyseal closure and slippage of the capital femoral epiphysis (Liu and LeRoith, 1999). A “slow-release” form of GH (from Genentech) has been developed that only requires injections every 14 days. However, this GH product appears to perturb the normal physiological pulsatile GH profile, and is also associated with frequent side effects.
In contrast, essentially no side effects have been reported for recombinant GHRH therapies. Extracranially secreted GHRH, as mature peptide or truncated molecules (as seen with pancreatic islet cell tumors and variously located carcinoids) are often biologically active and can even produce acromegaly (Esch et al., 1982; Thorner et al., 1984). Administration of recombinant GHRH to GH-deficient children or adult humans augments IGF-I levels, increases GH secretion proportionally to the GHRH dose, yet still invokes a response to bolus doses of recombinant GHRH (Bercu and Walker, 1997). Thus, GHRH administration represents a more physiological alternative of increasing subnormal GH and IGF-I levels (Corpas et al., 1993).
GH is released in a distinctive pulsatile pattern that has profound importance for its biological activity (Argente et al., 1996). Secretion of GH is stimulated by the GHRH, and inhibited by somatostatin, and both hypothalamic hormones (Thorner et al., 1995). GH pulses are a result of GHRH secretion that is associated with a diminution or withdrawal of somatostatin secretion. In addition, the pulse generator mechanism is timed by GH-negative feedback. Effective and regulated expression of the GH and IGF-I pathway is essential for optimal linear growth, homeostasis of carbohydrate, protein, and fat metabolism, and for providing a positive nitrogen balance (Murray and Shalet, 2000). Numerous studies in humans, sheep or pigs showed that continuous infusion with recombinant GHRH protein restores the normal GH pattern without desensitizing GHRH receptors or depleting GH supplies as this system is capable of feed-back regulation, which is abolished in the GH therapies (Dubreuil et al., 1990; Vance, 1990; Vance et al., 1985). Although recombinant GHRH protein therapy entrains and stimulates normal cyclical GH secretion with virtually no side effects, the short half-life of GHRH in vivo requires frequent (one to three times a day) intravenous, subcutaneous or intranasal (requiring 300-fold higher dose) administration. Thus, as a chronic treatment, GHRH administration is not practical.
Wild-type GHRH has a relatively short half-life in the circulatory system, both in humans (Frohman et al., 1984) and in farm animals. After 60 minutes of incubation in plasma, 95% of the GHRH(1-44)NH2 is degraded, while incubation of the shorter (1-40)OH form of the hormone under similar conditions, shows only a 77% degradation of the peptide (Frohman et al., 1989). Incorporation of cDNA coding for a particular protease-resistant GHRH analog in a therapeutic nucleic acid vector results in a molecule with a longer half-life in serum, increased potency, and provides greater GH release in plasmid-injected animals (Draghia-Akli et al., 1999), herein incorporated by reference. Mutagenesis via amino acid replacement of protease sensitive amino acids prolongs the serum half-life of the GHRH molecule. Furthermore, the enhancement of biological activity of GHRH is achieved by using super-active analogs that may increase its binding affinity to specific receptors (Draghia-Akli et al., 1999).
Growth Hormone (“GH”) and Growth Hormone Releasing Hormone (“GHRH”) in Farm animals: The administration of recombinant growth hormone (“GH”) or recombinant GHRH has been used in subjects for many years, but not as a pathway to treat arthritis, or to increase the arthritic patient welfare. More specifically, recombinant GH treatment in farm animals has been shown to enhance lean tissue deposition and/or milk production, while increasing feed efficiency (Etherton et al., 1986; Klindt et al., 1998). Numerous studies have shown that recombinant GH markedly reduces the amount of carcass fat and consequently the quality of products increases. However, chronic GH administration has practical, economical and physiological limitations that potentially mitigate its usefulness and effectiveness (Chung et al., 1985; Gopinath and Etherton, 1989b). Experimentally, recombinant GH-releasing hormone (“GHRH”) has been used as a more physiological alternative. The use of GHRH in large animal species (e.g. pigs or cattle) not only enhances growth performance and milk production, but more importantly, the efficiency of production from both a practical and metabolic perspective (Dubreuil et al., 1990; Farmer et al., 1992). For example, the use of recombinant GHRH in lactating sows has beneficial effects on growth of the weanling pigs, yet optimal nutritional and hormonal conditions are needed for GHRH to exert its full potential (Farmer et al., 1996). Administration of GHRH and GH stimulate milk production, with an increase in feed to milk conversion. This therapy enhances growth primarily by increasing lean body mass (Lapierre et al., 1991; van Rooij et al., 2000) with overall improvement in feed efficiency. Hot and chilled carcass weights are increased and carcass lipid (percent of soft-tissue mass) is decrease by administration of GHRH and GH (Etherton et al., 1986).
Transgene Delivery and in vivo Expression: Although not wanting to be bound by theory, the delivery of specific transgenes to somatic tissue to correct inborn or acquired deficiencies and imbalances is possible. Such transgene-based drug delivery offers a number of advantages over the administration of recombinant proteins. These advantages include: the conservation of native protein structure; improved biological activity; avoidance of systemic toxicities; and avoidance of infectious and toxic impurities. Because the protein is synthesized and secreted continuously into the circulation, plasmid mediated therapy allows for prolonged production of the protein in a therapeutic range. In contrast, the primary limitation of using recombinant protein is the limited availability of protein after each administration.
In a plasmid-based expression system, a non-viral transgene vector may comprise of a synthetic transgene delivery system in addition to the nucleic acid encoding the therapeutic genetic product. In this way, the risks associated with the use of most viral vectors can be avoided, including the expression of viral proteins that can induce immune responses against target tissues and the possibility of DNA mutations or activations of oncogenes. The non-viral expression vector products generally have low toxicity due to the use of “species-specific” components for gene delivery, which minimizes the risks of immunogenicity generally associated with viral vectors. Additionally, integration of plasmid sequences into host chromosomes is below the rate of spontaneous mutation, so that this type of nucleic acid vector therapy should neither activate oncogenes nor inactivate tumor suppressor genes (Ledwith et al., 2000b; Ledwith et al., 2000a). As episomal systems residing outside the chromosomes, plasmids have defined pharmacokinetics and elimination profiles, leading to a finite duration of gene expression in target tissues.
Direct plasmid DNA gene transfer is currently the basis of many emerging nucleic acid therapy strategies and does not require viral components or lipid particles (Aihara and Miyazaki, 1998; Muramatsu et al., 2001). Skeletal muscle is target tissue, because muscle fiber has a long life span and can be transduced by circular DNA plasmids that are expressed in immunocompetent hosts (Davis et al., 1993; Tripathy et al., 1996). Plasmid DNA constructs are attractive candidates for direct therapy into the subjects skeletal muscle because the constructs are well-defined entities that are biochemically stable and have been used successfully for many years (Acsadi et al., 1991; Wolff et al., 1990). The relatively low expression levels of an encoded product that are achieved after direct plasmid DNA injection are sometimes sufficient to indicate bio-activity of secreted peptides (Danko and Wolff, 1994; Tsurumi et al., 1996). Previous reports demonstrated that human GHRH cDNA could be delivered to muscle by an injectable myogenic expression vector in mice where it transiently stimulated GH secretion to a modest extent over a period of two weeks (Draghia-Akli et al., 1997).
There are several different approaches that can be utilized for the treatment of arthritis. Because arthritis is a chronic condition, effective treatment may require the presence of therapeutic agents for extended periods of time. In the case of proteins, this is problematic. Gene therapeutic approaches may offer a solution to this problem. Experimental studies have confirmed the feasibility, efficacy and safety of gene therapy for the treatment of animal models of arthritis. Several different approaches have shown promise in this regard, including gene transfer to the synovial lining cells of individual joints and the systemic delivery of genes to extra-articular locations. One unexpected finding has been the ‘contralateral effect’ in which gene delivery to one joint of an animal with poly-articular disease leads to improvement of multiple joints. Investigation of this phenomenon has led to interest in cell trafficking and the genetic modification of antigen-presenting cells (Gouze et al., 2001). Different types of molecules have been used. For instance, therapeutic strategies to block tumor necrosis factor alpha (TNF-alpha) activity in experimental autoimmune arthritis models and rheumatoid arthritis have proved highly successful, and provide sustained beneficial effects (Mukherjee et al., 2003); gene transfer of interleukin-1 receptor antagonist was also used as a treatment modality for the equine patients and offers future promise for human patients with osteoarthritis (Frisbie et al., 2002). Plasmid mediated GHRH supplementation that determines a reduction in TNF-alpha levels in dogs with spontaneous malignancies may act further through this route in the treatment of arthritis (Draghia-Akli et al., 2002a).
Efforts have been made to enhance the delivery of plasmid DNA to cells by physical means including electroporation, sonoporation, and pressure. Although not wanting to be bound by theory, the administration of a nucleic acid construct by electroporation involves the application of a pulsed electric field to create transient pores in the cellular membrane without causing permanent damage to the cell, which allows exogenous molecules to enter the cell (Smith and Nordstrom, 2000). By adjusting the electrical pulse generated by an electroporetic system, nucleic acid molecules can travel through passageways or pores in the cell that are created during the procedure. U.S. Pat. No. 5,704,908 titled “Electroporation and iontophoresis catheter with porous balloon,” issued on Jan. 6, 1998 with Hofmann et al., listed as inventors describes an electroporation apparatus for delivering molecules to cells at a selected location within a cavity in the body of a patient. Similar pulse voltage injection devices are also described in: U.S. Pat. No. 5,702,359 titled “Needle electrodes for mediated delivery of drugs and genes,” issued on Dec. 30, 1997, with Hofmann, et al., listed as inventors; U.S. Pat. No. 5,439,440 titled “Electroporation system with voltage control feedback for clinical applications,” issued on Aug. 8, 1995 with Hofmann listed as inventor; PCT application WO/96/12520 titled “Electroporetic Gene and Drug Therapy by Induced Electric Fields,” published on May 5, 1996 with Hofmann et al., listed as inventors; PCT application WO/96/12006 titled “Flow Through Electroporation Apparatus and Method,” published on Apr. 25, 1996 with Hofmann et al., listed as inventors; PCT application WO/95/19805 titled “Electroporation and Iontophoresis Apparatus and Method For insertion of Drugs and genes into Cells,” published on Jul. 27, 1995 with Hofmann listed as inventor; and PCT application WO/97/07826 titled “In Vivo Electroporation of Cells,” published on Mar. 6, 1997, with Nicolau et al., listed as inventors, the entire content of each of the above listed references is hereby incorporated by reference.
Recently, significant progress to enhance plasmid delivery in vivo and subsequently to achieve physiological levels of a secreted protein was obtained using the electroporation technique. Electroporation has been used very successfully to transfect tumor cells after injection of plasmid (Lucas et al., 2002; Matsubara et al., 2001)) or to deliver the anti-tumor drug bleomycin to cutaneous and subcutaneous tumors in humans (Gehl et al., 1998; Heller et al., 1996). Electroporation also has been extensively used in mice (Lesbordes et al., 2002; Lucas et al., 2001; Vilquin et al., 2001), rats (Terada et al., 2001; Yasui et al., 2001), and dogs (Fewell et al., 2001) to deliver therapeutic genes that encode for a variety of hormones, cytokines or enzymes. Previous studies using GHRH showed that plasmid therapy with electroporation is scalable and represents a promising approach to induce production and regulated secretion of proteins in large animals and humans (Draghia-Akli et al., 1999; Draghia-Akli et al., 2002c). Electroporation also has been extensively used in rodents and other small animals (Bettan et al., 2000; Yin and Tang, 2001). Intramuscular injection of plasmid followed by electroporation has been used successfully in ruminants for vaccination purposes (Babiuk et al., 2003; Tollefsen et al., 2003). It has been observed that the electrode configuration affects the electric field distribution, and subsequent results (Gehl et al., 1999; Miklavcic et al., 1998). Although not wanting to be bound by theory, needle electrodes give consistently better results than external caliper electrodes in a large animal model.
The ability of electroporation to enhance plasmid uptake into the skeletal muscle has been well documented. Similarly, plasmids formulated with poly-L-glutamate (“PLG”) or polyvinylpyrrolidone (“PVP”) were observed to have an increase in plasmid transfection, which consequently increased the expression of a desired transgene. For example, plasmids formulated with PLG or PVP were observed to increase gene expression to up to 10 fold in the skeletal muscle of mice, rats, and dogs (Fewell et al., 2001; Mumper et al., 1998). Although not wanting to be bound by theory, the anionic polymer sodium PLG enhances plasmid uptake at low plasmid concentrations and reduces any possible tissue damage caused by the procedure. PLG is a stable compound and it is resistant to relatively high temperatures (Dolnik et al., 1993). PLG has been used to increase stability of anti-cancer drugs (Li et al., 2000) and as “glue” to close wounds or to prevent bleeding from tissues during wound and tissue repair (Otani et al., 1996; Otani et al., 1998). PLG has been used to increase stability in vaccine preparations (Matsuo et al., 1994) without increasing their immunogenicity. PLG also has been used as an anti-toxin after antigen inhalation or exposure to ozone (Fryer and Jacoby, 1993).
Although not wanting to be bound by theory, PLG increases the transfection of the plasmid during the electroporation process, not only by stabilizing the plasmid DNA and facilitating the intracellular transport through the membrane pores, but also through an active mechanism. For example, positively charged surface proteins on the cells could complex the negatively charged PLG linked to plasmid DNA through protein-protein interactions. When an electric field is applied, the surface proteins reverse direction and actively internalize the DNA molecules, a process that substantially increases the transfection efficiency. Furthermore, PLG will prevent the muscle damage associated with in vivo plasmid delivery (Draghia-Akli et al., 2002b) and will increase plasmid stability in vitro prior to injection. There are studies directed to electroporation of eukaryotic cells with linear DNA (McNally et al., 1988; Neumann et al., 1982) (Toneguzzo et al., 1988) (Aratani et al., 1992; Nairn et al., 1993; Xie and Tsong, 1993; Yorifuji and Mikawa, 1990), but these examples illustrate transfection into cell suspensions, cell cultures, and the like, and such transfected cells are not present in a somatic tissue.
U.S. Pat. No. 4,956,288 is directed to methods for preparing recombinant host cells containing high copy number of a foreign DNA by electroporating a population of cells in the presence of the foreign DNA, culturing the cells, and killing the cells having a low copy number of the foreign DNA.
Although not wanting to be bound by theory, a GHRH cDNA can be delivered to muscle of mice and humans by an injectable myogenic expression vector where it can transiently stimulate GH secretion over a period of two weeks (Draghia-Akli et al., 1997). This injectable vector system was optimized by incorporating a powerful synthetic muscle promoter (Li et al., 1999) coupled with a novel protease-resistant GHRH molecule with a substantially longer half-life and greater GH secretory activity (pSP-HV-GHRH) (Draghia-Akli et al., 1999). Highly efficient electroporation technology was optimized to deliver the nucleic acid construct to the skeletal muscle of an animal (Draghia-Akli et al., 2002b). Using this combination of vector design and electric pulses plasmid delivery method, the inventors were able to show increased growth and favorably modified body composition in pigs (Draghia-Akli et al., 1999; Draghia-Akli et al., 2003) and rodents (Draghia-Akli et al., 2002c). The modified GHRH nucleic acid constructs increased red blood cell production in companion animals with cancer and cancer treatment-associated anemia (Draghia-Akli et al., 2002a). In pigs, available data suggested that the modified porcine HV-GHRH analog (SEQID# 1) was more potent in promoting growth and positive body composition changes than the wild-type porcine GHRH (Draghia-Akli et al., 1999).
Administering novel GHRH analog proteins (U.S. Pat. Nos. 5,847,066; 5846,936; 5,792,747; 5,776,901; 5,696,089; 5,486,505; 5,137,872; 5,084,442, 5,036,045; 5,023,322; 4,839,344; 4,410,512, RE33,699) or synthetic or naturally occurring peptide fragments of GHRH (U.S. Pat. Nos. 4,833,166; 4,228,158; 4,228,156; 4,226,857; 4,224,316; 4,223,021; 4,223,020; 4,223,019) for the purpose of increasing release of growth hormone have been reported. A GHRH analog containing the following mutations have been reported (U.S. Pat. No. 5,846,936): Tyr at position 1 to His; Ala at position 2 to Val, Leu, or others; Asn at position 8 to Gln, Ser, or Thr; Gly at position 15 to Ala or Leu; Met at position 27 to Nle or Leu; and Ser at position 28 to Asn. The GHRH analog is the subject of U.S. Pat. No. 6,551,996 titled “Super-active porcine growth hormone releasing hormone analog,” issued on Apr. 22, 2003 with Schwartz, et al., listed as inventors (“the '996 Patent”), which teaches application of a GHRH analog containing mutations that improve the ability to elicit the release of growth hormone. In addition, the '996 Patent application relates to the treatment of growth deficiencies; the improvement of growth performance; the stimulation of production of growth hormone in an animal at a greater level than that associated with normal growth; and the enhancement of growth utilizing the administration of growth hormone releasing hormone analog and is herein incorporated by reference.
U.S. Pat. No. 5,874,534 (“the '534 patent”) and U.S. Pat. No. 5,935,934 (“the '934 patent”) describe mutated steroid receptors, methods for their use and a molecular switch for nucleic acid vector therapy, the entire content of each is hereby incorporated by reference. A molecular switch for regulating expression in nucleic acid vector therapy and methods of employing the molecular switch in humans, animals, transgenic animals and plants (e.g. GeneSwitch®) are described in the '534 patent and the '934 patent. The molecular switch is described as a method for regulating expression of a heterologous nucleic acid cassette for nucleic acid vector therapy and is comprised of a modified steroid receptor that includes a natural steroid receptor DNA binding domain attached to a modified ligand binding domain. The modified binding domain usually binds only non-natural ligands, anti-hormones or non-native ligands. One skilled in the art readily recognizes natural ligands do not readily bind the modified ligand-binding domain and consequently have very little, if any, influence on the regulation or expression of the gene contained in the nucleic acid cassette.
In summary preventing or treating arthritis, and preventing and treating lameness particularly in animals, and improving the welfare of an arthritic subject were previously uneconomical and restricted in scope. The related art has shown that it is possible to improve these different conditions in a limited capacity utilizing recombinant protein technology, but these treatments have some significant drawbacks. It has also been taught that nucleic acid expression constructs that encode recombinant proteins are viable solutions to the problems of frequent injections and high cost of traditional recombinant therapy. There is a need in the art to expanded treatments for subjects with a disease by utilizing nucleic acid expression constructs that are delivered into a subject and express stable therapeutic proteins in vivo.